Avian cells have been used for years for the production of viral vaccines. For instance, embryonated chick eggs are able to support the replication of a wide range of human and animal viruses including notably attenuated viruses which have impaired potential to replicate in human or mammalian cells. Embryonated chick eggs used for human viral vaccine production must be certified to be free of a defined set of viral and bacterial contamination (specific pathogen-free or SPF). Embryonated chick eggs are expensive and can constitute up to 40% of the cost of viral vaccines. A vaccine lot cannot be released until the SPF supplier verifies that the parental chickens for the embryonated chick eggs used to manufacture the vaccine lot were completely free of any disease. This uncertainty adds a significant cost to the preparation of these vaccines. In addition, the large-scale processes for infecting eggs and maintaining virus growth are time consuming and sometimes inconsistent across different vaccine batches.
With the development of cell culture techniques vaccine manufacturers have replaced embryonated chick eggs with isolated chicken embryo fibroblasts (CEFs). Most Flaviviridae vaccines are for instance manufactured from CEFs. The Flaviviridae Yellow fever viral vaccines recommended by the World Health Organization (WHO) are from 17D strain and substrains, which have minor differences in nucleotide sequences and equivalent immunogenicity (CAMACHO L. A. B. et al., Rev Saude Publica 38(5):671-8 (2004); DOS SANTOS C. N., Virus Res. 35:35-41 (1995)). Experiments regarding Flaviviridae viral production process have resulted in the establishment of an efficient methodology for the production of a Yellow fever viral vaccine in CEFs using the 17DD virus strain (FREIRE M. S., Vaccine 23, 2501-2512 (2005)). While the use of CEFs improves the safety profile, efficiency and reliability of the manufacturing process, it also highly time consuming and further increases costs. The production of CEFs depends indeed on the availability of SPF eggs. The CEFs are prepared from SPF eggs by mincing embryos to establish and amplify viable cells. Typical for primary animal cells the fibroblasts suffer senescence: the doubling time increases with passaging and eventually all cells die. This process occurs after about 20 passages. This limited live span constraints to perform a complete set of safety tests for each lot of CEFs.
Due to the limitations of embryonated chicken eggs and CEFs as previously described for manufacturing viral vaccines, the search for new cell culture systems have drawn considerable attention over the past decades. In this respect immortalized cell lines are highly attractive. Immortalized cell lines can be maintained or frozen from batch to batch on the production site and are always available for a new production process. Moreover as they are confined at the production plant, they are less subject to contamination by exogenous contaminant. Their use allows a drastic reduction of the manual manipulation needed for the production process. All these properties lead to a reduction of the price and of the duration of the production process as well as a diminution of the potential contamination. Immortalized cell lines can moreover be fully characterized and are thus totally compliant with the good laboratory practice and the requirements of the different medical agencies. As an example, VERO cell line has been described as alternative candidate for Flaviviridae vaccine production in replacement of the approved embryonated chicken eggs. The live attenuated Japanese encephalitis virus vaccine ChimeriVax™-JEV was propagated in VERO cells cultured in media supplemented with fetal bovine serum (MONATH et al., Biologicals, 33:131-144 (2005)). Similarly, MATEU et al. (Trans. R. Soc. Trop. Med. Hyg., 101(3):289-98 (2007)) and TORINIWA et al. (Vaccine, 4; 26(29-30):3680-9 (2008)) have respectively produced Yellow fever virus vaccine and Japanese encephalitis virus vaccine in VERO cell line. The application of immortalized cell lines is moreover not limited to the viral vaccines they are designed for but may be extended to recombinant proteins. For example, both antibodies and influenza viral vaccines can be propagated on PER.C6® cell line (ECACC number 96022940), a human fetal retinoblast cell line immortalized by transfection with an E1 minigene of adeno virus type 5. (FALLAUX F. J. et al., Hum. Gene Ther. 9:1909-17 (1998); PAU M. G. et al., Vaccine 19, 2716-2721 (2001); YALLOP C. et al., Animal Cell Technology meets Genomics, 533-536 (2005)).
However alternative immortalized cell lines are still needed. WO2007/077256 provides methods for immortalizing primary avian cells by transfection with the Cairina moschata telomerase reverse transcriptase (TERT) nucleic acid molecule either by targeted or random insertion of the TERT nucleic acid molecule into the Cairina moschata primary avian cells genome. Based on said methods, especially by random insertion, we have now identified new immortalized avian cell lines which have distinguished properties from one another.